Nucleotide sequences which code for the atr43 gene

ABSTRACT

The invention relates to an isolated polynucleotide having a polynucleotide sequence which codes for the atr43 gene, and a host-vector system having a coryneform host bacterium in which the atr43 gene is present in attenuated form and a vector which carries at least the atr43 gene according to SEQ ID No 1, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

BACKGROUND OF THE INVENTION

[0001] The invention provides nucleotide sequences from coryneformbacteria which code for the atr43 gene and a process for thefermentative preparation of amino acids using bacteria in which theatr43 gene is attenuated. All references cited herein are expresslyincorporated by reference. Incorporation by reference is also designatedby the term “I.B.R.” following any citation.

[0002] L-Amino acids, in particular L-lysine, are used in human medicineand in the pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

[0003] It is known that amino acids are prepared by fermentation fromstrains of coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocess can relate to fermentation measures, such as, for example,stirring and supply of oxygen, or the composition of the nutrient media,such as, for example, the sugar concentration during the fermentation,or the working up to the product form by, for example, ion exchangechromatography, or the intrinsic output properties of the microorganismitself.

[0004] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and which produce amino acids are obtained in thismanner.

[0005] Methods of the recombinant DNA technique have also been employedfor some years for improving the strain of Corynebacterium strains whichproduce L-amino acid, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

[0006] The inventors had the object of providing new measures forimproved fermentative preparation of amino acids.

[0007] BRIEF SUMMARY OF THE INVENTION

[0008] Where L-amino acids or amino acids are mentioned in thefollowing, this means one or more amino acids, including their salts,chosen from the group consisting of L-asparagine, L-threonine, L-serine,L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,L-lysine, L-tryptophan and L-arginine. L-Lysine is particularlypreferred.

[0009] When L-lysine or lysine are mentioned in the following, not onlythe bases but also the salts, such as e.g. lysine monohydrochloride orlysine sulfate, are meant by this.

[0010] The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence which codes for the atr43gene, chosen from the group consisting of

[0011] a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence of SEQ ID No. 2,

[0012] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is identical to the extent of at least 70%to the amino acid sequence of SEQ ID No. 2,

[0013] c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0014] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c),

[0015] the polypeptide preferably having the activity of the ABCtransporter Atr43.

[0016] The invention also provides the above-mentioned polynucleotide,this preferably being a DNA which is capable of replication, comprising:(i) the nucleotide sequence, shown in SEQ ID No. 1, or (ii) at least onesequence which corresponds to sequence (i) within the range of thedegeneration of the genetic code, or (iii) at least one sequence whichhybridizes with the sequences complementary to sequences (i) or (ii),and optionally (iv) sense mutations of neutral function in (i).

[0017] The invention also provides:

[0018] a polynucleotide, in particular DNA, which is capable ofreplication and comprises the nucleotide sequence as shown in SEQ IDNo.1;

[0019] a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence as shown in SEQ ID No. 2;

[0020] a vector containing parts of the polynucleotide according to theinvention, but at least 15 successive nucleotides of the sequenceclaimed,

[0021] and coryneform bacteria in which the atr43 gene is attenuated, inparticular by an insertion or deletion.

[0022] The invention also provides polynucleotides which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library of a coryneformbacterium, which comprises the complete gene or parts thereof, with aprobe which comprises the sequence of the polynucleotide according tothe invention according to SEQ ID No.1 or a fragment thereof, andisolation of the polynucleotide sequence mentioned.

BRIEF DESCRIPTION OF THE FIGURE

[0023]FIG. 1 is a map of the plasmid pCR2.latr43int.

[0024] The abbreviations and designations used have the followingmeaning. KmR: Kanamycin resistance gene KpnI: Cleavage site of therestriction enzyme KpnI EcoRI: Cleavage site of the restriction enzymeEcoRI PstI: Cleavage site of the restriction enzyme PstI atr43int:Internal fragment of the atr43 gene ColE1: Replication origin of theplasmid ColE1

DETAILED DESCRIPTION OF THE INVENTION

[0025] Polynucleotides which comprise the sequences according to theinvention are suitable as hybridization probes for RNA, cDNA and DNA, inorder to isolate, in the full length, nucleic acids or polynucleotidesor genes which code for the ABC transporter Atr43 or to isolate thosenucleic acids or polynucleotides or genes which have a high similaritywith the sequence of the atr43 gene. They are also suitable forincorporation into so-called “arrays”, “micro arrays” or “DNA chips” inorder to detect and determine the corresponding polynucleotides.

[0026] Polynucleotides which comprise the sequences according to theinvention are furthermore suitable as primers with the aid of which DNAof genes which code for the ABC transporter Atr43 can be prepared by thepolymerase chain reaction (PCR).

[0027] Such oligonucleotides which serve as probes or primers compriseat least 30, preferably at least 20, very particularly preferably atleast 15 successive nucleotides. Oligonucleotides which have a length ofat least 40 or 50 nucleotides are also suitable. Oligonucleotides with alength of at least 100, 150, 200, 250 or 300 nucleotides are optionallyalso suitable.

[0028] “Isolated” means separated out of its natural environment.

[0029] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0030] The polynucleotides according to the invention include apolynucleotide according to SEQ ID No. 1 or a fragment preparedtherefrom and also those which are at least 70% to 80%, preferably atleast 81% to 85%, particularly preferably at least 86% to 90%, and veryparticularly preferably at least 91%, 93%, 95%, 97% or 99% identical tothe polynucleotide according to SEQ ID No. 1 or a fragment preparedtherefrom.

[0031] “Polypeptides” are understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0032] The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of the ABC transporter Atr43, and also those which are at least70% to 80%, preferably at least 81% to 85%, particularly preferably atleast 91%, 93%, 95%, 97% or 99% identical to the polypeptide accordingto SEQ ID No. 2 and have the activity mentioned.

[0033] The invention furthermore relates to a process for thefermentative preparation of amino acids chosen from the group consistingof L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine,L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan andL-arginine using coryneform bacteria which in particular already produceamino acids and in which the nucleotide sequences which code for theatr43 gene are attenuated, in particular eliminated or expressed at alow level.

[0034] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

[0035] By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to10% or 0 to 5% of the activity or concentration of the wild-typeprotein.

[0036] The microorganisms to which the present invention relates canprepare amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

[0037] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum (C. glutamicum), are inparticular the known wild-type strains

[0038]Corynebacterium glutamicum ATCC13032

[0039]Corynebacterium acetoglutamicum ATCC15806

[0040]Corynebacterium acetoacidophilum ATCC13870

[0041]Corynebacterium melassecola ATCC17965

[0042]Corynebacterium thermoaminogenes FERM BP-1539

[0043]Brevibacterium flavum ATCC14067

[0044]Brevibacterium lactofermentum ATCC13869 and

[0045]Brevibacterium divaricatum ATCC14020

[0046] and L-amino acid-producing mutants or strains prepared therefrom.

[0047] The new atr43 gene from C. glutamicum which codes for the ABCtransporter Atr43 has been isolated.

[0048] To isolate the atr43 gene or also other genes of C. glutamicum, agene library of this microorganism is first set up in Escherichia coli(E. coli). The setting up of gene libraries is described in generallyknown textbooks and handbooks. The textbook by Winnacker: Gene undKlone, Eine Einführung in die Gentechnologie [Genes and Clones, AnIntroduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany,1990 I.B.R.), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989 I.B.R.) maybe mentioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50,495-508 (1987)) I.B.R. Bathe et al. (Molecular and General Genetics,252:255-265, 1996) I.B.R. describe a gene library of C. glutamicumATCC13032, which was set up with the aid of the cosmid vector SuperCos I(Wahl et al., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164 I.B.R.) in the E. coli K-12 strain NM554 (Raleigh et al.,1988, Nucleic Acids Research 16:1563-1575 I.B.R.).

[0049] Bormann et al. (Molecular Microbiology 6(3), 317-326 (1992))I.B.R. in turn describe a gene library of C. glutamicum ATCC13032 usingthe cosmid pHC79 (Hohn and Collins, 1980, Gene 11, 291-298 I.B.R.).

[0050] To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences,25, 807-818 I.B.R.) or pUC9 (Vieira et al., 1982, Gene, 19:259-268I.B.R.). Suitable hosts are, in particular, those E. coli strains whichare restriction- and recombination-defective, such as, for example, thestrain DH5αmcr, which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649) I.B.R. Thelong DNA fragments cloned with the aid of cosmids or other λ vectors canthen in turn be subcloned and subsequently sequenced in the usualvectors which are suitable for DNA sequencing, such as is described e.g.by Sanger et al. (Proceedings of the National Academy of Sciences of theUnited States of America, 74:5463-5467, 1977) I.B.R.

[0051] The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)) I.B.R., that of Marck(Nucleic Acids Research 16, 1829-1836 (1988)) I.B.R. or the GCG programof Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) I.B.R.

[0052] The new DNA sequence of C. glutamicum which codes for the atr43gene and which, as SEQ ID No. 1, is a constituent of the presentinvention has been found. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of theatr43 gene product is shown in SEQ ID No. 2.

[0053] Coding DNA sequences which result from SEQ ID No. 1 by thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Conservativeamino acid exchanges, such as e.g. exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. It isfurthermore known that changes on the N and/or C terminus of a proteincannot substantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)) I.B.R.,in O'Regan et al. (Gene 77:237-251 (1989)) I.B.R., in Sahin-Toth et al.(Protein Sciences 3:240-247 (1994)) I.B.R., in Hochuli et al.(Bio/Technology 6:1321-1325 (1988)) I.B.R. and in known textbooks ofgenetics and molecular biology. Amino acid sequences which result in acorresponding manner from SEQ ID No. 2 are also a constituent of theinvention.

[0054] In the same way, DNA sequences which hybridize with SEQ ID No. 1or parts of SEQ ID No. 1 are a constituent of the invention. Finally,DNA sequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

[0055] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” from BoehringerMannheim GmbH (Mannheim, Germany, 1993) I.B.R. and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991))I.B.R. The hybridization takes place under stringent conditions, that isto say only hybrids in which the probe and target sequence, i.e. thepolynucleotides treated with the probe, are at least 70% identical areformed. It is known that the stringency of the hybridization, includingthe washing steps, is influenced or determined by varying the buffercomposition, the temperature and the salt concentration. Thehybridization reaction is preferably carried out under a relatively lowstringency compared with the washing steps (Hybaid Hybridisation Guide,Hybaid Limited, Teddington, UK, 1996) I.B.R.

[0056] A 5× SSC buffer at a temperature of approx. 50° C.-68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides which are less than 70% identical tothe sequence of the probe. Such hybrids are less stable and are removedby washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2× SSC and optionallysubsequently 0.5× SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995 I.B.R.) atemperature of approx. 50° C. -68° C. being established. It isoptionally possible to lower the salt concentration to 0.1× SSC.Polynucleotide fragments which are, for example, at least 70% or atleast 80% or at least 90% to 95% identical to the sequence of the probeemployed can be isolated by increasing the hybridization temperaturestepwise from 50° C. to 68° C. in steps of approx. 1-2° C. Furtherinstructions on hybridization are obtainable on the market in the formof so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH,Mannheim, Germany, Catalogue No. 1603558).

[0057] Instructions for amplification of DNA sequences with the aid ofthe polymerase chain reaction (PCR) can be found by the expert, interalia, in the handbook by Gait: Oligonucleotide Synthesis: A PracticalApproach (IRL Press, Oxford, UK, 1984) I.B.R. and in Newton and Graham:PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) I.B.R.

[0058] It has been found that coryneform bacteria produce amino acids inan improved manner after attenuation of the atr43 gene.

[0059] To achieve an attenuation, either the expression of the atr43gene or the catalytic properties of the enzyme protein can be reduced oreliminated. The two measures can optionally be combined.

[0060] The reduction in gene expression can take place by suitableculturing or by genetic modification (mutation) of the signal structuresof gene expression. Signal structures of gene expression are, forexample, repressor genes, activator genes, operators, promoters,attenuators, ribosome binding sites, the start codon and terminators.The expert can find information on this e.g. in the patent applicationWO 96/15246 I.B.R., in Boyd and Murphy (Journal of Bacteriology 170:5949 (1988)) I.B.R., in Voskuil and Chambliss (Nucleic Acids Research26: 3548 (1998) I.B.R., in Jensen and Hammer (Biotechnology andBioengineering 58: 191 (1998)) I.B.R., in Pátek et al. (Microbiology142: 1297 (1996)) I.B.R., Vasicova et al. (Journal of Bacteriology 181:6188 (1999)) I.B.R. and in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik[Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, 1995) I.B.R. or that by Winnacker (“Gene und Klone [Genes andClones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R.

[0061] Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)) I.B.R., Sugimoto et al.(Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) I.B.R.and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum:Aufhebung der allosterischen Regulation und Struktur des Enzyms[Threonine dehydratase from Corynebacterium glutamicum: Canceling theallosteric regulation and structure of the enzyme]”, Reports from theJulich Research Center, Jül -2906, ISSN09442952, Jüilich, Germany, 1994)I.B.R. Summarizing descriptions can be found in known textbooks ofgenetics and molecular biology, such as e.g. that by Hagemann(“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, 1986) I.B.R.

[0062] Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair (bp) in agene lead to frame shift mutations, as a consequence of which incorrectamino acids are incorporated or translation is interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik[Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, 1995) I.B.R., that by Winnacker (“Gene und Klone [Genes andClones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R. orthat by Hagemann (“Allgemeine Genetik [General Genetics]”, GustavFischer Verlag, Stuttgart, 1986) I.B.R.

[0063] A common method of mutating genes of C. glutamicum is the methodof “gene disruption” and “gene replacement” described by Schwarzer andPuhler (Bio/Technology 9, 84-87 (1991)) I.B.R.

[0064] In the method of gene disruption a central part of the codingregion of the gene of interest is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983) I.B.R.), pK18mob or pK19mob (Schafer et al., Gene 145,69-73 (1994) I.B.R.), pK18mobsacB or pK19mobsacB (Jäger et al., Journalof Bacteriology 174: 5462-65 (1992) I.B.R.), pGEM-T (Promegacorporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal ofBiological Chemistry 269:32678-84 I.B.R.; U.S. Pat. No. 5,487,993I.B.R.), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al.,Journal of Molecular Biology, 234: 534-541 (1993) I.B.R.) or pEM1(Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516 I.B.R.).The plasmid vector which contains the central part of the coding regionof the gene is then transferred into the desired strain of C. glutamicumby conjugation or transformation. The method of conjugation isdescribed, for example, by Schafer et al. (Applied and EnvironmentalMicrobiology 60, 756-759 (1994) I.B.R.). Methods for transformation aredescribed, for example, by Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)) I.B.R., Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989) I.B.R.) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994) I.B.R.). After homologousrecombination by means of a “cross-over” event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used, for example, by Fitzpatrick etal. (Applied Microbiology and Biotechnology 42, 575-580 (1994) I.B.R.)to eliminate the recA gene of C. glutamicum.

[0065] In the method of “gene replacement”, a mutation, such as e.g. adeletion, insertion or base exchange, is established in vitro in thegene of interest. The allele prepared is in turn cloned in a vectorwhich is not replicative for C. glutamicum and this is then transferredinto the desired host of C. glutamicum by transformation or conjugation.After homologous recombination by means of a first “cross-over” eventwhich effects integration and a suitable second “cross-over” event whicheffects excision in the target gene or in the target sequence, theincorporation of the mutation or of the allele is achieved. This methodwas used, for example, by Peters-Wendisch et al. (Microbiology 144,915-927 (1998)) I.B.R. to eliminate the pyc gene of C. glutamicum by adeletion.

[0066] A deletion, insertion or a base exchange can be incorporated intothe atr43 gene in this manner.

[0067] In addition, it may be advantageous for the production of L-aminoacids to enhance, in particular over-express, one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the attenuation ofthe atr43 gene.

[0068] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures.

[0069] By enhancement measures, in particular over-expression, theactivity or concentration of the corresponding protein is in generalincreased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, up to a maximum of 1000% or 2000%, based on the startingmicroorganism.

[0070] Thus, for the preparation of L-amino acids, in addition toattenuation of the atr43 gene, at the same time one or more of the geneschosen from the group consisting of

[0071] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335 I.B.R.),

[0072] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086I.B.R.),

[0073] the tpi gene which codes for triose phosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086

[0074] the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086 I.B.R.),

[0075] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-09224661 I.B.R.),

[0076] the pyc gene which codes for pyruvate carboxylase (DE-A-198 31609 I.B.R.),

[0077] the mqo gene which codes for malate-quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)I.B.R.),

[0078] the lysC gene which codes for a feed-back resistant aspartatekinase (Accession No.P26512; EP-B-0387527 I.B.R.; EP-A-0699759 I.B.R.),

[0079] the lysE gene which codes for lysine export (DE-A-195 48 222I.B.R.),

[0080] the hom gene which codes for homoserine dehydrogenase (EP-A0131171 I.B.R.),

[0081] the ilvA gene which codes for threonine dehydratase (Mockel etal., Journal of Bacteriology (1992) 8065-8072) I.B.R.) or the ilvA(Fbr)allele which codes for a “feed back resistant” threonine dehydratase(Möckel et al., (1994) Molecular Microbiology 13: 833-842 I.B.R.),

[0082] the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B0356739 I.B.R.),

[0083] the ilvD gene which codes for dihydroxy-acid dehydratase (Sahmand Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979I.B.R.),

[0084] the zwa1 gene which codes for the Zwa1 protein (DE: 19959328.0I.B.R., DSM 13115)

[0085] can be enhanced, in particular over-expressed.

[0086] It may furthermore be advantageous for the production of aminoacids, in addition to attenuation of the atr43 gene, at the same timefor one or more of the genes chosen from the group consisting of

[0087] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1 I.B.R., DSM 13047),

[0088] the pgi gene which codes for glucose 6-phosphate isomerase (U.S.Ser. No. 09/396,478 I.B.R., DSM 12969),

[0089] the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7I.B.R., DSM 13114),

[0090] the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2I.B.R., DSM 13113)

[0091] to be attenuated, in particular for the expression thereof to bereduced.

[0092] In addition to the attenuation of the atr43 gene it mayfurthermore be advantageous for the production of amino acids toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982I.B.R.).

[0093] The invention also provides the microorganisms prepared accordingto the invention, and these can be cultured continuously ordiscontinuously in the batch process (batch culture) or in the fed batch(feed process) or repeated fed batch process (repetitive feed process)for the purpose of production of L-amino acids. A summary of knownculture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991) I.B.R.) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen [Bioreactors and PeripheralEquipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) I.B.R.

[0094] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981) I.B.R.

[0095] Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas, for example, soya oil, sunflower oil, groundnut oil and coconut fat,fatty acids, such as, for example, palmitic acid, stearic acid andlinoleic acid, alcohols, such as, for example, glycerol and ethanol, andorganic acids, such as, for example, acetic acid, can be used as thesource of carbon. These substances can be used individually or as amixture.

[0096] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0097] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as, for example, magnesium sulfate oriron sulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the above-mentioned substances. Suitable precursors canmoreover be added to the culture medium. The starting substancesmentioned can be added to the culture in the form of a single batch, orcan be fed in during the culturing in a suitable manner.

[0098] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture. Antifoams, such as, for example, fatty acid polyglycolesters, can be employed to control the development of foam. Suitablesubstances having a selective action, such as, for example, antibiotics,can be added to the medium to maintain the stability of plasmids. Tomaintain aerobic conditions, oxygen or oxygen-containing gas mixtures,such as, for example, air, are introduced into the culture. Thetemperature of the culture is usually 20° C. to 45° C., and preferably25° C. to 40° C. Culturing is continued until a maximum of the desiredproduct has formed. This target is usually reached within 10 hours to160 hours.

[0099] Methods for the determination of L-amino acids are known from theprior art. The analysis can thus be carried out, for example, asdescribed by Spackman et al. (Analytical Chemistry, 30, (1958), 1190)I.B.R. by anion exchange chromatography with subsequent ninhydrinderivation, or it can be carried out by reversed phase HPLC, for exampleas described by Lindroth et al. (Analytical Chemistry (1979) 51:1167-1174) I.B.R.

[0100] The process according to the invention is used for thefermentative preparation of L-amino acids, in particular of L-lysine.The amino acids are in general isolated by conventional processes orseparated off together with constituents of the fermentation broth andoptionally the entire biomass or portions thereof.

[0101] The following microorganism was deposited as a pure culture onApr. 11, 2001 at the Deutsche Sammlung fur Mikroorganismen undZellkulturen (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

[0102]Escherichia coli Top10/pCR2.1atr43int as DSM 14226.

[0103] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

[0104] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatmentwere carried out by the method of Sambrook et al. (Molecular Cloning. ALaboratory Manual, 1989, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA) I.B.R. Methods for transformation ofEscherichia coli are also described in this handbook.

[0105] The composition of the usual nutrient media, such as LB or TYmedium, can also be found in the handbook by Sambrook et al.

EXAMPLE 1

[0106] Preparation of a Genomic Cosmid Gene Library from C. glutamicumATCC 13032

[0107] Chromosomal DNA from C. glutamicum ATCC₁₃₀₃₂ was isolated asdescribed by Tauch et al. (1995, Plasmid 33:168-179 I.B.R.) and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02I.B.R.). The DNA fragments were dephosphorylated with shrimp alkalinephosphatase (Roche Molecular Biochemicals, Mannheim, Germany, ProductDescription SAP, Code no. 1758250 I.B.R.). The DNA of the cosmid vectorSuperCos1 (Wahl et al. (1987), Proceedings of the National Academy ofSciences, USA 84:2160-2164 I.B.R.), obtained from Stratagene (La Jolla,USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301I.B.R.) was cleaved with the restriction enzyme XbaI (AmershamPharmacia, Freiburg, Germany, Product Description XbaI, Code no.27-0948-02 I.B.R.) and likewise dephosphorylated with shrimp alkalinephosphatase.

[0108] The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04 I.B.R.). The cosmid DNA treated in this manner was mixedwith the treated ATCC13032 DNA and the batch was treated with T4 DNAligase (Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04 I.B.R.). The ligation mixture was thenpacked in phages with the aid of Gigapack II XL Packing Extracts(Stratagene, La Jolla, USA, Product Description Gigapack II XL PackingExtract, Code no. 200217 I.B.R.).

[0109] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Res. 16:1563-1575 I.B.R.) the cells were taken up in 10 mMMgSO₄ and mixed with an aliquot of the phage suspension. The infectionand titering of the cosmid library were carried out as described bySambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor) I.B.R., the cells being plated out on LB agar (Lennox,1955, Virology, 1:190 I.B.R.)+100 μg/ml ampicillin. After incubationovernight at 37° C., recombinant individual clones were selected.

EXAMPLE 2

[0110] Isolation and Sequencing of the atr43 Gene

[0111] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02 I.B.R.). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, Product DescriptionSAP, Product No. 1758250 I.B.R.). After separation by gelelectrophoresis, the cosmid fragments in the size range of 1500 to 2000bp were isolated with the QiaExII Gel Extraction Kit (Product No. 20021,Qiagen, Hilden, Germany).

[0112] The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, Holland, Product Description Zero BackgroundCloning Kit, Product No. K2500-01 I.B.R.) was cleaved with therestriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, ProductDescription BamHI, Product No. 27-0868-04 I.B.R.). The ligation of thecosmid fragments in the sequencing vector pZero-1 was carried out asdescribed by Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor I.B.R.), the DNA mixture being incubatedovernight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). Thisligation mixture was then electroporated (Tauch et al. 1994, FEMSMicrobiol. Letters, 123:343-7 I.B.R.) into the E. coli strain DH5αmcr(Grant, 1990, Proceedings of the National Academy of Sciences, U.S.A.,87:4645-4649 I.B.R.) and plated out on LB agar (Lennox, 1955, Virology,1:190 I.B.R.) with 50 μg/ml zeocin.

[0113] The plasmid preparation of the recombinant clones was carried outwith the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany).The sequencing was carried out by the dideoxy chain termination methodof Sanger et al. (1977, Proceedings of the National Academies ofSciences, U.S.A., 74:5463-5467 I.B.R.) with modifications according toZimmermann et al. (1990, Nucleic Acids Research, 18:1067) I.B.R. The “RRdRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems(Product No. 403044, Weiterstadt, Germany I.B.R.) was used. Theseparation by gel electrophoresis and analysis of the sequencingreaction were carried out in a “Rotiphoresis NFAcrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

[0114] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231 I.B.R.)version 97-0. The individual sequences of the pzerol derivatives wereassembled to a continuous contig. The computer-assisted coding regionanalyses were prepared with the XNIP program (Staden, 1986, NucleicAcids Research, 14:217-231 I.B.R.). Further analyses were carried outwith the “BLAST search programs” (Altschul et al., 1997, Nucleic AcidsResearch, 25:3389-3402 I.B.R.) against the non-redundant databank of the“National Center for Biotechnology Information” (NCBI, Bethesda, Md.,USA I.B.R.).

[0115] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of the nucleotide sequence showed an open reading frame of 1626bp, which was called the atr43 gene. The atr43 gene codes for apolypeptide of 541 amino acids.

EXAMPLE 3

[0116] Preparation of an Integration Vector for Integration Mutagenesisof the atr43 Gene

[0117] From the strain ATCC₁₃₀₃₂, chromosomal DNA was isolated by themethod of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) I.B.R. Onthe basis of the sequence of the atr43 gene known for C. glutamicum fromexample 2, the following oligonucleotides were chosen for the polymerasechain reaction: atr43-int1: 5′ GCA GCT TAA AAC CCT GTC C 3′ (SEQ IDNO:3) atr43-int2: 5′ GTT GTC GAT CAT TCG TTC C 3′ (SEQ ID NO:4)

[0118] The primers shown were synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out by the standard PCR methodof Innis et al. (PCR protocols. A guide to methods and applications,1990, Academic Press I.B.R.) with the Taq-polymerase from BoehringerMannheim (Germany, Product Description Taq DNA polymerase, Product No. 1146 165). With the aid of the polymerase chain reaction, the primersallow amplification of an internal fragment of the atr43 gene 460 bp insize. The product amplified in this way was tested electrophoreticallyin a 0.8% agarose gel.

[0119] The amplified DNA fragment was ligated with the TOPO TA CloningKit from Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue NumberK4500-01) in the vector pCR2.1-TOPO (Mead at al. (1991) Bio/Technology9:657-663 I.B.R.).

[0120] The E. coli strain TOP10 was then electroporated with theligation batch (Hanahan, In: DNA cloning. A practical approach. Vol. I,IRL-Press, Oxford, Washington DC, USA, 1985 I.B.R.). Selection ofplasmid-carrying cells was carried out by plating out the transformationbatch on LB Agar (Sambrook et al., Molecular cloning: a laboratorymanual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 I.B.R.), which had been supplemented with 50 mg/lkanamycin. Plasmid DNA was isolated from a transformant with the aid ofthe QIAprep Spin Miniprep Kit from Qiagen and checked by restrictionwith the restriction enzyme EcoRI and subsequent agarose gelelectrophoresis (0.8%). The plasmid was called pCR2.1atr43int and isshown in FIG. 1.

EXAMPLE 4 Integration Mutagenesis of the atr43 Gene in the Strain DSM

[0121] The vector pCR2.1atr43int mentioned in example 3 waselectroporated by the electroporation method of Tauch et al.(FEMSMicrobiological Letters, 123:343-347 (1994)) I.B.R. in Corynebacteriumglutamicum DSM 5715. The strain DSM 5715 is an AEC-resistant lysineproducer(EP 435 132 I.B.R.). The vector pCR2.1atr43int cannot replicateindependently in DSM5715 and is retained in the cell only if it hasintegrated into the chromosome of DSM 5715.

[0122] Selection of clones with pCR2.1atr43int integrated into thechromosome was carried out by plating out the electroporation batch onLB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2^(nd)Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.I.B.R.), which had been supplemented with 15 mg/l kanamycin.

[0123] For detection of the integration, the atr43int fragment waslabeled with the Dig hybridization kit from Boehringer by the method of“The DIG System Users Guide for Filter Hybridization” of BoehringerMannheim GmbH (Mannheim, Germany, 1993 I.B.R.). Chromosomal DNA of apotential integrant was isolated by the method of Eikmanns et al.(Microbiology 140: 1817-1828 (1994)) I.B.R. and in each case cleavedwith the restriction enzymes KpnI, EcoRI and PstI. The fragments formedwere separated by means of agarose gel electrophoresis and hybridized at68° C. with the Dig hybridization kit from Boehringer. The plasmidpCR2.1atr43int mentioned in example 3 had been inserted into thechromosome of DSM5715 within the chromosomal atr43 gene. The strain wascalled DSM5715::pCR2.1atr43int.

EXAMPLE 5

[0124] Preparation of Lysine

[0125] The C. glutamicum strain DSM5715::pCR2.1atr43int obtained inexample 4 was cultured in a nutrient medium suitable for the productionof lysine and the lysine content in the culture supernatant wasdetermined.

[0126] For this, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with kanamycin (25 mg/l))for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the preculture. MediumCg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/lGlucose (autoclaved separately) 2% (w/v) The pH was brought to pH 7.4

[0127] Kanamycin (25 mg/l) was added to this. The preculture wasincubated for 16 hours at 33° C. at 240 rpm on a shaking machine. A mainculture was seeded from this preculture such that the initial OD (660nm) of the main culture was 0.1 OD. Medium MM was used for the mainculture. Medium MM CSL (corn steep liquor) 5 g/l MOPS(morpholinopropanesulfonic acid) 20 g/l Glucose (autoclaved separately)50 g/l Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/lCaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lLeucine (sterile-filtered) 0.1 g/l CaCO₃ 25 g/l

[0128] The CSL, MOPS and the salt solution are brought to pH 7 withaqueous ammonia and autoclaved. The sterile substrate and vitaminsolutions are then added, and the CaCO₃ autoclaved in the dry state isadded.

[0129] Culturing was carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added. Culturing was carriedout at 33° C. and 80% atmospheric humidity.

[0130] After 72 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of lysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivation with ninhydrin detection.

[0131] The result of the experiment is shown in Table 1. TABLE 1 ODLysine HCl Strain (660 nm) g/l DSM5715 8.7 12.64 DSM5715::pCR2.latr43int9.1 13.57

[0132] This application claims priority to German Priority DocumentApplication No. 100 45 580.8, filed on Sep. 15, 2000 and to GermanPriority Document Application No. 101 23 070.2, filed on May 11, 2001.Both German Priority Documents are hereby incorporated by reference intheir entirety.

1 4 1 2080 DNA Corynebacterium glutamicum CDS (249)..(1871) 1 aagaggaacccaccagcacc gaagaacaac cgtcagaaac tgagcagcct tctgaacctg 60 aagaggaatcgactggtgtt gctggaagct ctaacggtgg cagttttgtc gcgcttttag 120 cgctgctggcagcgcttggt ggcatcgtcg gtgcagtcct cggattgctt aagttgtagg 180 tggctgggggcgtcgaaaag cagctttatt gacctggcaa cttcaattga tagactgtta 240 ggttgtga ttgtca cca atg att aga ggt gcg cgt tgg cgc acg tac ctt 290 Met Ser Pro MetIle Arg Gly Ala Arg Trp Arg Thr Tyr Leu 1 5 10 ctc gat gcc cca ggt cagctc ctt cgg gtg cag cca ggc gac cgt att 338 Leu Asp Ala Pro Gly Gln LeuLeu Arg Val Gln Pro Gly Asp Arg Ile 15 20 25 30 ggt ctg gtt ggt aga aatggt gcg ggc aaa acc acc acc atg cga atc 386 Gly Leu Val Gly Arg Asn GlyAla Gly Lys Thr Thr Thr Met Arg Ile 35 40 45 ctc tcg ggc gaa acc aag ccctac gga gga tcc gta acc aca tct ggt 434 Leu Ser Gly Glu Thr Lys Pro TyrGly Gly Ser Val Thr Thr Ser Gly 50 55 60 gaa atc ggt tac ctg ccc cag gactcc cgc gaa ggc aac atc gaa caa 482 Glu Ile Gly Tyr Leu Pro Gln Asp SerArg Glu Gly Asn Ile Glu Gln 65 70 75 acc gcc cgc gac cga gtc ctc tcc gcccgt ggt ctt gac cag ctg cga 530 Thr Ala Arg Asp Arg Val Leu Ser Ala ArgGly Leu Asp Gln Leu Arg 80 85 90 tcc tcc atg gaa cgc cag cag gag atc atggaa acc gcg acc gac ccc 578 Ser Ser Met Glu Arg Gln Gln Glu Ile Met GluThr Ala Thr Asp Pro 95 100 105 110 ggc aag ctc gac gcc gcg atc cgc aagtac tcc agg ctc gag gaa caa 626 Gly Lys Leu Asp Ala Ala Ile Arg Lys TyrSer Arg Leu Glu Glu Gln 115 120 125 ttc cag tcg ctc ggc ggc tac gaa gctgac gcc gaa gca gcc cag atc 674 Phe Gln Ser Leu Gly Gly Tyr Glu Ala AspAla Glu Ala Ala Gln Ile 130 135 140 tgc gac aac ctc ggc ctc gag gca cgcatc ctc gac cag cag ctt aaa 722 Cys Asp Asn Leu Gly Leu Glu Ala Arg IleLeu Asp Gln Gln Leu Lys 145 150 155 acc ctg tcc ggc ggc cag cgc cgc cgcgtc gag ttg gcg cag atc ctc 770 Thr Leu Ser Gly Gly Gln Arg Arg Arg ValGlu Leu Ala Gln Ile Leu 160 165 170 ttc gcc gcc acc aac ggc tcc ggc aaatca aaa acc aca ttg ctt ctc 818 Phe Ala Ala Thr Asn Gly Ser Gly Lys SerLys Thr Thr Leu Leu Leu 175 180 185 190 gac gag ccc acc aac cac ttg gacgca gac tcg atc acc tgg ctc cgt 866 Asp Glu Pro Thr Asn His Leu Asp AlaAsp Ser Ile Thr Trp Leu Arg 195 200 205 gac ttc ctg gcg aag cac gaa ggtgga ctg atc atg att tcg cac gac 914 Asp Phe Leu Ala Lys His Glu Gly GlyLeu Ile Met Ile Ser His Asp 210 215 220 gtc gaa ctg ctt ggc gcc gta tgtaac aag att tgg tac ctc gac gca 962 Val Glu Leu Leu Gly Ala Val Cys AsnLys Ile Trp Tyr Leu Asp Ala 225 230 235 gta cgc agc gaa gcc gat gtc tacaac atg ggc ttt agc aaa tac gtc 1010 Val Arg Ser Glu Ala Asp Val Tyr AsnMet Gly Phe Ser Lys Tyr Val 240 245 250 gat gca cgt gca ctc gat gaa gcacgc cga cgc cgt gag cgc gca aac 1058 Asp Ala Arg Ala Leu Asp Glu Ala ArgArg Arg Arg Glu Arg Ala Asn 255 260 265 270 gcc gaa aag aag gcc gga gccctc aag gac cag gct gca cgc ctc ggc 1106 Ala Glu Lys Lys Ala Gly Ala LeuLys Asp Gln Ala Ala Arg Leu Gly 275 280 285 gcg aaa gca acc aag gct gccgca gct aag cag atg atc gcc cgt gcg 1154 Ala Lys Ala Thr Lys Ala Ala AlaAla Lys Gln Met Ile Ala Arg Ala 290 295 300 gaa cga atg atc gac aac ctcgac gaa atc cgc gta gct gac cgc gcc 1202 Glu Arg Met Ile Asp Asn Leu AspGlu Ile Arg Val Ala Asp Arg Ala 305 310 315 gcc aac atc gtt ttc cca gaacca gca ccc tgt gga aaa acc cca ctc 1250 Ala Asn Ile Val Phe Pro Glu ProAla Pro Cys Gly Lys Thr Pro Leu 320 325 330 aac gcc aag ggc ctg acc aagatg tac ggc tcc ctc gaa gtc ttc gcc 1298 Asn Ala Lys Gly Leu Thr Lys MetTyr Gly Ser Leu Glu Val Phe Ala 335 340 345 350 ggc gtc gac cta gcc atcgac aaa ggc tcc cgc gta gtc gtc ctc gga 1346 Gly Val Asp Leu Ala Ile AspLys Gly Ser Arg Val Val Val Leu Gly 355 360 365 ttc aac ggt gca ggt aaaacc acc ctg ctc aaa ctc ctc gcc ggt gtg 1394 Phe Asn Gly Ala Gly Lys ThrThr Leu Leu Lys Leu Leu Ala Gly Val 370 375 380 gaa cgc acc gac ggc gaaggc ggc atc gtc acc gga tac ggc ctc aaa 1442 Glu Arg Thr Asp Gly Glu GlyGly Ile Val Thr Gly Tyr Gly Leu Lys 385 390 395 atc ggc tac ttc gcc caggaa cac gac acc atc gac ccc gac aaa tcc 1490 Ile Gly Tyr Phe Ala Gln GluHis Asp Thr Ile Asp Pro Asp Lys Ser 400 405 410 gtc tgg caa aac acc atcgaa gcc tgc gcc gac gcc gac caa caa agc 1538 Val Trp Gln Asn Thr Ile GluAla Cys Ala Asp Ala Asp Gln Gln Ser 415 420 425 430 ctc cgc agc ctc ctcgga tcc ttc atg ttc tcc ggc gaa caa ctc gac 1586 Leu Arg Ser Leu Leu GlySer Phe Met Phe Ser Gly Glu Gln Leu Asp 435 440 445 caa cca gca gga acactc tcc ggc ggt gaa aaa acc cgc ctc gca ctg 1634 Gln Pro Ala Gly Thr LeuSer Gly Gly Glu Lys Thr Arg Leu Ala Leu 450 455 460 gcc acc ctc gtg tcctcc cgc gca aac gtc ctg ctt ctc gac gag ccc 1682 Ala Thr Leu Val Ser SerArg Ala Asn Val Leu Leu Leu Asp Glu Pro 465 470 475 acc aac aac ctt gacccg atc tcc cgc gaa cag gtc ctc gac gca ctg 1730 Thr Asn Asn Leu Asp ProIle Ser Arg Glu Gln Val Leu Asp Ala Leu 480 485 490 cgc acc tac acc ggcgca gtc gtc ctg gtt acc cac gac ccg ggt gca 1778 Arg Thr Tyr Thr Gly AlaVal Val Leu Val Thr His Asp Pro Gly Ala 495 500 505 510 gtc aag gcc cttgag cca gaa cgc gtc atc gtg ctt cct gat ggc acc 1826 Val Lys Ala Leu GluPro Glu Arg Val Ile Val Leu Pro Asp Gly Thr 515 520 525 gag gat ctt tggaat gat cag tac atg gaa atc gtg gaa ttg gcg 1871 Glu Asp Leu Trp Asn AspGln Tyr Met Glu Ile Val Glu Leu Ala 530 535 540 taggttctaa ggctgtttatgctggtcaag actgtttcgt tttaaactcc tgcacttagc 1931 cgcccgcaag ttgcccgcatgaatttcatc atgcgggcta tttcagctgc tggctgattg 1991 ttgaggccag ggaagaaggggacataagtg tccaaggttg tcgataccgt cgtatgcccc 2051 aaatctctct gaacctgcttcaccgagac 2080 2 541 PRT Corynebacterium glutamicum 2 Met Ser Pro MetIle Arg Gly Ala Arg Trp Arg Thr Tyr Leu Leu Asp 1 5 10 15 Ala Pro GlyGln Leu Leu Arg Val Gln Pro Gly Asp Arg Ile Gly Leu 20 25 30 Val Gly ArgAsn Gly Ala Gly Lys Thr Thr Thr Met Arg Ile Leu Ser 35 40 45 Gly Glu ThrLys Pro Tyr Gly Gly Ser Val Thr Thr Ser Gly Glu Ile 50 55 60 Gly Tyr LeuPro Gln Asp Ser Arg Glu Gly Asn Ile Glu Gln Thr Ala 65 70 75 80 Arg AspArg Val Leu Ser Ala Arg Gly Leu Asp Gln Leu Arg Ser Ser 85 90 95 Met GluArg Gln Gln Glu Ile Met Glu Thr Ala Thr Asp Pro Gly Lys 100 105 110 LeuAsp Ala Ala Ile Arg Lys Tyr Ser Arg Leu Glu Glu Gln Phe Gln 115 120 125Ser Leu Gly Gly Tyr Glu Ala Asp Ala Glu Ala Ala Gln Ile Cys Asp 130 135140 Asn Leu Gly Leu Glu Ala Arg Ile Leu Asp Gln Gln Leu Lys Thr Leu 145150 155 160 Ser Gly Gly Gln Arg Arg Arg Val Glu Leu Ala Gln Ile Leu PheAla 165 170 175 Ala Thr Asn Gly Ser Gly Lys Ser Lys Thr Thr Leu Leu LeuAsp Glu 180 185 190 Pro Thr Asn His Leu Asp Ala Asp Ser Ile Thr Trp LeuArg Asp Phe 195 200 205 Leu Ala Lys His Glu Gly Gly Leu Ile Met Ile SerHis Asp Val Glu 210 215 220 Leu Leu Gly Ala Val Cys Asn Lys Ile Trp TyrLeu Asp Ala Val Arg 225 230 235 240 Ser Glu Ala Asp Val Tyr Asn Met GlyPhe Ser Lys Tyr Val Asp Ala 245 250 255 Arg Ala Leu Asp Glu Ala Arg ArgArg Arg Glu Arg Ala Asn Ala Glu 260 265 270 Lys Lys Ala Gly Ala Leu LysAsp Gln Ala Ala Arg Leu Gly Ala Lys 275 280 285 Ala Thr Lys Ala Ala AlaAla Lys Gln Met Ile Ala Arg Ala Glu Arg 290 295 300 Met Ile Asp Asn LeuAsp Glu Ile Arg Val Ala Asp Arg Ala Ala Asn 305 310 315 320 Ile Val PhePro Glu Pro Ala Pro Cys Gly Lys Thr Pro Leu Asn Ala 325 330 335 Lys GlyLeu Thr Lys Met Tyr Gly Ser Leu Glu Val Phe Ala Gly Val 340 345 350 AspLeu Ala Ile Asp Lys Gly Ser Arg Val Val Val Leu Gly Phe Asn 355 360 365Gly Ala Gly Lys Thr Thr Leu Leu Lys Leu Leu Ala Gly Val Glu Arg 370 375380 Thr Asp Gly Glu Gly Gly Ile Val Thr Gly Tyr Gly Leu Lys Ile Gly 385390 395 400 Tyr Phe Ala Gln Glu His Asp Thr Ile Asp Pro Asp Lys Ser ValTrp 405 410 415 Gln Asn Thr Ile Glu Ala Cys Ala Asp Ala Asp Gln Gln SerLeu Arg 420 425 430 Ser Leu Leu Gly Ser Phe Met Phe Ser Gly Glu Gln LeuAsp Gln Pro 435 440 445 Ala Gly Thr Leu Ser Gly Gly Glu Lys Thr Arg LeuAla Leu Ala Thr 450 455 460 Leu Val Ser Ser Arg Ala Asn Val Leu Leu LeuAsp Glu Pro Thr Asn 465 470 475 480 Asn Leu Asp Pro Ile Ser Arg Glu GlnVal Leu Asp Ala Leu Arg Thr 485 490 495 Tyr Thr Gly Ala Val Val Leu ValThr His Asp Pro Gly Ala Val Lys 500 505 510 Ala Leu Glu Pro Glu Arg ValIle Val Leu Pro Asp Gly Thr Glu Asp 515 520 525 Leu Trp Asn Asp Gln TyrMet Glu Ile Val Glu Leu Ala 530 535 540 3 19 DNA Corynebacteriumglutamicum 3 gcagcttaaa accctgtcc 19 4 19 DNA Corynebacterium glutamicum4 gttgtcgatc attcgttcc 19

We claim:
 1. An isolated polynucleotide from coryneform bacteria,comprising a polynucleotide sequence which codes for the atr43 gene,selected from the group consisting of a) a polynucleotide which isidentical to the extent of at least 70% to a polynucleotide which codesfor a polypeptide which comprises the amino acid sequence of SEQ ID No.2, b) a polynucleotide which codes for a polypeptide which comprises anamino acid sequence which is identical to the extent of at least 70% tothe amino acid sequence of SEQ ID No. 2, c) a polynucleotide which iscomplementary to the polynucleotides of a) or b), and d) apolynucleotide comprising at least 15 successive nucleotides of thepolynucleotide sequence of a), b) or c),
 2. The polynucleotide accordingto claim 1, wherein the polypeptide of a) or b) has the activity of theABC transporter Atr43.
 3. The polynucleotide according to claim 1,wherein the polynucleotide is a recombinant DNA which is capable ofreplication in coryneform bacteria.
 4. The polynucleotide according toclaim 1, wherein the polynucleotide is an RNA.
 5. The polynucleotideaccording to claim 3, comprising the nucleic acid sequence as shown inSEQ ID No.
 1. 6. The polynucleotide according to claim 3, whereinpolynucleotide is a DNA which is capable of replication, comprising (i)the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least onesequence which corresponds to sequence (i) within the range of thedegeneration of the genetic code, or (iii) at least one sequence whichhybridizes with the sequence complementary to sequence (i) or (ii). 7.The polynucleotide according to claim 6, further comprising (iv) sensemutations of neutral function in (i).
 8. The polynucleotide according toclaim 6, wherein the hybridization is carried out under a stringencycorresponding to at most 2× SSC.
 9. The polynucleotide sequenceaccording to claim 1, which codes for a polypeptide which comprises theamino acid sequence shown in SEQ ID No.
 2. 10. A coryneform bacteria inwhich the atr43 gene is attenuated
 11. A coryneform bacteria in whichthe atr43 gene is eliminated.
 12. The Coryneform bacteria, according toclaim 11; wherein the atr43 gene is eliminated by integrationmutagenesis.
 13. An integration vector pCR2.latr43int, deposited in thestrain DSM
 14226. 14. A method for the fermentative preparation ofL-amino acids in coryneform bacteria comprising: a) fermenting, in amedium, the coryneform bacteria which produce the desired L-amino acidand in which at least the atr43 gene or nucleotide sequences which codefor it are attenuated.
 15. The method according to claim 14, furthercomprising: b) concentrating the L-amino acid in the medium or in thecells of the bacteria.
 16. The method according to claim 15, furthercomprising: c) isolating the L-amino acid.
 17. The method according toclaim 16, wherein constituents of a fermentation broth or an entirebiomass are present.
 18. The method according to claim 14, wherein theL-amino acids are L-lysine.
 19. The method according to claim 14,wherein at least the atr43 gene or nucleotide sequences which code forit are eliminated
 20. The method according to claim 14, wherein bacteriain which further genes of the biosynthesis pathway of the desiredL-amino acid are additionally enhanced are employed.
 21. The methodaccording to claim 14, wherein bacteria in which the metabolic pathwayswhich reduce the formation of the desired L-amino acid are at leastpartly eliminated are employed.
 22. The method according to claim 14,wherein the expression of the polynucleotide(s) which code(s) for theatr43 gene is attenuated.
 23. The method according to claim 14, whereinthe expression of the polynucleotide(s) which code(s) for the atr43 geneis eliminated.
 24. The method according to claim 14, wherein thecatalytic properties of the polypeptide, for which the polynucleotideatr43 codes, are reduced.
 25. The method according to claim 20, whereinthe bacteria being fermented comprise, at the same time, one or moregenes which are enhanced; wherein the one or more genes is/are selectedfrom the group consisting of: the dapA gene which codes fordihydrodipicolinate synthase, the gap gene which codes forglyceraldehyde 3-phosphate dehydrogenase, the tpi gene which codes fortriose phosphate isomerase, the pgk gene which codes for3-phosphoglycerate kinase, the zwf gene which codes for glucose6-phosphate dehydrogenase, the pyc gene which codes for pyruvatecarboxylase, the mqo gene which codes for malate-quinone oxidoreductase,the lysC gene which codes for a feed-back resistant aspartate kinase,the lysE gene which codes for lysine export, the hom gene which codesfor homoserine dehydrogenase the ilvA gene which codes for threoninedehydratase or the ilvA(Fbr) allele which codes for a feed backresistant threonine dehydratase, the ilvBN gene which codes foracetohydroxy-acid synthase, the ilvD gene which codes for dihydroxy-aciddehydratase, and the zwa1 gene which codes for the Zwa1 protein.
 26. Themethod according to claim 25, wherein the one or more genes areoverexpressed.
 27. The method according to claim 22, wherein thebacteria being fermented comprise, at the same time, one or more geneswhich are attenuated; wherein the one or more genes is/are selected fromthe group consisting of: the pgi gene which codes for glucose6-phosphate isomerase, the poxB gene which codes for pyruvate oxidase,and the zwa2 gene which codes for the Zwa2 protein.
 28. The methodaccording to claim 14, wherein microorganisms of the genusCorynebacterium glutamicum are employed.
 29. A coryneform bacteriacomprising a vector which carries parts of the polynucleotide accordingto claim 1, but at least 15 successive nucleotides of the sequenceclaimed.
 30. A method for discovering RNA, cDNA and DNA in order toisolate nucleic acids or polynucleotides or genes which code for the ABCtransporter Atr43 or have a high similarity with the sequence of theatr43 gene, comprising contacting the RNA, CDNA, or DNA withhybridization probes comprising polynucleotide sequences according toclaim
 1. 31. The method according to claim 30, wherein arrays, microarrays or DNA chips are employed.